Modified Mareks Disease Virus, and Vaccines Made Therefrom

ABSTRACT

The present invention provides an effective vaccine for Marek&#39;s disease, which may be prepared using a recombinant Marek&#39;s Disease Virus (MDV), strain CVI988, having been transformed with a foreign DNA construct that includes a long terminal repeat sequence of a reticuloendotheliosis virus. This safe viral agent elicits a highly protective immune response in a chicken against virulent MDV challenge without causing a significant degree of pathogenicity. Suitable formulations of the vaccine for use in chickens include an effective immunization dosage of this novel viral agent, along with a pharmaceutically acceptable carrier or diluent.

INCORPORATION BY REFERENCE

This application claims priority to U.S. provisional patent applicationNo. 61/614,142, which was filed on Mar. 22, 2012, and is hereinincorporated by reference in its entirety.

FIELD OF THE INVENTION

The present invention relates generally to viral vaccines and methods ofusing the same. More particularly, the present invention relates tonovel vaccines for protecting chickens against infection with Marek'sdisease virus, and having improved safety and efficacy over existingvaccines.

BACKGROUND

Marek's disease (MD), a highly prevalent and importantlymphoproliferative disease of chickens, is controlled in commercialchickens by live virus vaccines consisting of attenuated or naturallyavirulent MD-related herpesviruses. Although vaccination programs havebeen considered effective overall, the poultry industry continues toexperience losses due to MD. Given the tendency of MD virus to becomemore virulent with time (e.g. by reversion to more virulent form)coupled with the economic pressures confronting the poultry industry,there remains a strong incentive to develop safer and more efficaciousproducts that will protect better in the face of early challenge withvery virulent field strains without causing adverse side effects (e.g.thymic dystrophy).

There are three distinct serotypes of MD virus found in chickens: (1)serotype 1, the oncogenic form responsible for the disease, includinghigh- and low-virulence MD virus and their attenuated variants; (2)serotype 2, a non-oncogenic MD virus; and (3) serotype 3, herpesvirus ofturkeys (HVT). An early MD vaccine consists of the serotype 3 virusoriginally isolated from turkeys as reported in Witter et al. [Am. J.vet. Res. 31:525-538 (1970)] and Okazaki et al. [U.S. Pat. No.3,642,574]. Its lack of oncogenicity, self-limiting infection, goodreplication in vivo and in vitro, availability as cell-free andcell-associated preparations, and high protective efficacy haveestablished HVT as a standard for WID vaccines throughout the world. Acommonly used strain of HVT is FC126.

Vaccines produced from the naturally avirulent SB-1 strain [Schat etal., J. Natl. Cancer inst. 60:1075-1082 (1978) and U.S. Pat. No.4,160,0241, an isolate of a serotype 2 MD virus] have been licensed inthe united States since 1984. The SB-1 strain is poorly protectiveagainst the highly virulent MDV strains. It is usually used incombination with HVT as a bivalent vaccine since the two virusestogether produce greater protection than does either one alone [Schat eta/., Avian Pathol. 11:593-606 (1982); Witter, Avian Pathol. 11:49-62(1982), the contents of which are incorporated by reference herein].This phenomenon has been termed “protective synergism.” The SB-1+HVTbivalent vaccine represents greater than 50% of the United States marketfor MD vaccines at present and is considered to be among the mostefficacious of the various MD products available. However, sporadiclosses occur despite its use.

Another MD vaccine produced from strain CVI988 clone C (CVI988/C) hasbeen licensed for commercial use in the United States. This vaccine wasderived from a mildly virulent serotype 1 MD virus attenuated by serialpassage in tissue culture and has been reported by De Boer et al. [AvianDis. 30:276-283 (1986)]. A further passaged derivative of CVI988/C,identified as CVI988/C/R6, has also been described by De Boer et al.[Advances in Marek's Disease Research, pp. 405-4 3 (1988)]. Morerecently, the original low-passage strain, designated CVI988/Rispens,which has been in commercial use in other countries for a number ofyears, was found to be highly effective against challenge with severalvery virulent MD virus strains by Witter et al. [4th Intl. Symp. Marek'sDisease, pp. 315-319 (1992)].

An experimental vaccine derived from Md11, a very virulent serotype 1 MDfield isolate, was reported by Witter, supra. Md11 was attenuated byserially passaging 75 times in cell culture, and the resultant vaccinewas designated Md11/75C. This vaccine has been shown to provide goodprotection against challenge with Md5 and most other highly virulent MDviruses tested; but it was less efficacious against challenge with theJM/102W strain, a prototype MD virus effectively protected against byHVT and SB-1 vaccines. Furthermore, its efficacy was consistently lowerin chicks with HVT antibody.

U.S. Pat. No. 4,895,717, Witter disclosed a revertant derivative ofMdn/75C which was referred to as Md11/75C/R2. Md11/75C/R2 was shown tobe superior to several other monovalent vaccines and was the equal of abivalent (HVT+SB-1) vaccine [Witter, Avian Dis. 31:752-765 (1987)].However, the inherent pathogenicity of serotype 1 viruses and thepotential of attenuated strains to revert to greater pathogenicity[Witter et al., Avian Pathol. 13:75-92 (1984)] are factors to beconsidered in the licensing of such products. A clone derived fromfurther passages of the Mdn/75C/R2 strain, designated Md11/75C/R2/23 (orR2/23), was found by Witter et al. [Avian Dis., 35:877-891 (1991)] topossess the highly protective nature of the parent strain without itsresidual pathogenicity.

Witter also described another MD vaccine derived from 301 B/1, anonpathogenic serotype 2 field isolate, in U.S. Pat. No. 4,895,718, thecontents of which are incorporated by reference herein, strain 301 B/1possessed superior replicative ability to SB-1, as well as greaterprotectivity against challenge to viruses.

A recombinant Marek's disease virus, referred to as RM1, having the longterminal repeats of reticuloendotheliosis virus stably integrated intothe repeat short (RS) regions of its genome was also described. Thisstrain was generated at the USDA-ARS-ADOL from a pathogenic serotype 1Marek's disease virus strain JM [Witter et al., 1997, Avian Dis.,41:407-421, and Jones et al., 1996, J. Virology, 70(4):2460-2467i.However, while the RM1 strain has been shown to provide a level ofprotection similar or superior to that of CVI988, it has also beenassociated with residual pathogenicity, causing thymic atrophy intreated birds.

Thus, although existing HVT, SB-1, CVI988, CVI988/C, Md11/75C,Md11/75C/R2 and 301 B/1 all elicit immune responses against certain MDviruses, none of these vaccines protect optimally against all MDchallenge viruses in all chickens. Moreover, these vaccines haveexhibited reduced efficacy against some of the more recently isolatedvery virulent strains of MD virus. To avert any large-scale outbreaks ofMD in the future, the need exists to develop safer vaccines havingimproved efficacy against highly virulent strains of MD virus.

SUMMARY OF THE INVENTION

The present invention is based, in part, upon the production and use ofvaccines comprising Marek's Disease Viruses (MDV) originally disclosedin application number U.S. Ser. No. 10/623,891 (published asUS2005/0019348A1, to Reddy et al.), the contents of which, as well asthe application's entire prosecution history, are both hereinincorporated by reference in their entirety. Specifically, the presentinvention stems from the surprising and unexpected finding that theCVRM2 “virus” (MDV strain “CVI988” transformed with a foreign DNAconstruct; disclosed, for example, in Table 1 of Reddy et al.) was notin fact a clonally distinct, single recombinant attenuated Marek'sDisease Virus (MDV). Instead, Applicants determined the CVRM2 was aheterogeneous population of recombinant and parental MDV, and when theyisolated and subsequently administered to avians a pure clonal line ofCVRM2 (hereinafter referred to as “RN1250”), they obtained safety andefficacy results far exceeding those a skilled person would haveexpected on reading Reddy et al.

In accordance with this discovery, it is an object of the invention toprovide a novel, highly protective vaccine against MD in avians,including chickens. It is also an object of the invention to provide avaccine which provides greater protection against highly virulentstrains of Marek's disease virus than those vaccines presently incommercial use.

It is another object of the invention to improve the viability andproductivity of chickens, particularly broilers and layers, and toreduce economic losses in the poultry industry caused by Marek'sdisease.

These and other embodiments are disclosed or are obvious from, andencompassed by, the following Detailed Description.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows a schematic organization of MDV genome, which contains aunique long (UL) region flanked by inverted repeat (IRS), terminalrepeat long (TRL), internal repeat long (IRL), and a unique short region(US), and is flanked by two inverted repeats, internal repeat short(IRS) and terminal repeat short (TRS). Also shown is a schematicrepresentation of the overlapping cosmid clones generated to rescue aninfectious virus from a highly virulent strain of MDV;

FIG. 2 depicts generation of the B40-Pac cosmid used to generate theCVRM vaccine;

FIG. 3 illustrates PCR-based diagnostic of recombinant MDV; presentedare the PCR primers, expected product sizes, and agarose gel image,which indicates the CVRM2 was not clonal, but in fact a dual populationof recombinant and parental MDV. Lanes: 1) ladder; 2) negative; 3)Rispens; 4) GA 22; 5) Rismavac; 6) RB1B; 7) RN1250; 8) positive(original mixed population);

FIG. 4 presents PCR confirmation of RN1250 MSV, RN1250 x+5, and BP5.Lanes: no template (1), RN1250 MSV (2), RN1250 x+5 (3), RN1250 BP5 (4).PCR reactions with all primer pairs resulted in the expected PCR productand banding pattern, thus there was no evidence for presence of theparental Rispens virus among RN1250 MSV, RN1250 x+5, and BP5 isolates.

DETAILED DESCRIPTION OF THE INVENTION Definitions

An “immunological response” to a composition or vaccine is thedevelopment in the host of a cellular and/or antibody-mediated immuneresponse to a composition or vaccine of interest. Usually, an“immunological response” includes but is not limited to one or more ofthe following effects: the production of antibodies, B cells, helper Tcells, and/or cytotoxic T cells, directed specifically to an antigen orantigens included in the composition or vaccine of interest. Preferably,the host will display either a therapeutic or protective immunologicalresponse such that resistance to new infection will be enhanced and/orthe clinical severity of the disease reduced. Such protection will bedemonstrated by either a reduction or lack of symptoms normallydisplayed by an infected host, a quicker recovery time and/or a loweredviral titer in the infected host.

By “animal” is intended mammals, birds, and the like. Animal or host asused herein includes mammals and human. The animal may be selected fromthe group consisting of equine (e.g., horse), canine (e.g., dogs,wolves, foxes, coyotes, jackals), feline (e.g., lions, tigers, domesticcats, wild cats, other big cats, and other felines including cheetahsand lynx), ovine (e.g., sheep), bovine (e.g., cattle), porcine (e.g.,pig), avian (e.g., chicken, duck, goose, turkey, quail, pheasant,parrot, finches, hawk, crow, ostrich, emu and cassowary), primate (e.g.,prosimian, tarsier, monkey, gibbon, ape), ferrets, seals, and fish. Theterm “animal” also includes an individual animal in all stages ofdevelopment, including embryonic and fetal stages.

Unless otherwise explained, all technical and scientific terms usedherein have the same meaning as commonly understood by one of ordinaryskill in the art to which this disclosure belongs. The singular terms“a”, “an”, and “the” include plural referents unless context clearlyindicates otherwise. Similarly, the word “or” is intended to include“and” unless the context clearly indicate otherwise.

It is noted that in this disclosure and particularly in the claimsand/or paragraphs, terms such as “comprises”, “comprised”, “comprising”and the like can have the meaning attributed to it in U.S. Patent law;e.g., they can mean “includes”, “included”, “including”, and the like;and that terms such as “consisting essentially of” and “consistsessentially of” have the meaning ascribed to them in U.S. Patent law,e.g., they allow for elements not explicitly recited, but excludeelements that are found in the prior art or that affect a basic or novelcharacteristic of the invention.

Cloning. The selection and propagation of (a) genetic material from asingle individual, (b) a vector containing one gene or gene fragment, or(c) a single organism containing one such gene or gene fragment.

Cloning vector. A plasmid, virus, retrovirus, bacteriophage, cosmid,artificial chromosome (bacterial or yeast), or nucleic acid sequencewhich is able to replicate in a host cell, characterized by one or asmall number of restriction endonuclease recognition sites at which thesequence may be cut in a predetermined fashion, and which may contain anoptional marker suitable for use in the identification. of transformedcells, e.g., tetracycline resistance or ampicillin resistance. A cloningvector may or may not possess the features necessary for it to operateas an expression vector.

Expression. The process undergone by a structural gene to produce apolypeptide. Expression requires transcription of DNA,post-transcriptional modification of the initial RNA transcript, andtranslation of RNA.

Expression Cassette. A nucleic acid sequence within a vector which is tobe transcribed, and a promoter to direct the transcription. Theexpression cassette may contain one or more unrelated DNA sequencesencoding one or more peptides of interest.

Expression Control Sequence. Expression control sequences are DNAsequences involved in any way in the control of transcription ortranslation and must include a promoter. Suitable expression controlsequences and methods of making and using them are well known in theart.

Expression vector. A replicon such as a plasmid, virus, retrovirus,bacteriophage, cosmid, artificial chromosome (bacterial or yeast), ornucleic acid sequence which is able to replicate in a host cell,characterized by a restriction endonuclease recognition site at whichthe sequence may be cut in a predetermined fashion for the insertion ofa heterologous DNA sequence. An expression vector has a promoterpositioned upstream of the site at which the sequence is cut for theinsertion of the heterologous DNA sequence, the recognition site beingselected so that the promoter will be operatively associated with theheterologous D{umlaut over (ι)} A sequence. A heterologous DNA sequenceis “operatively associated” with the promoter in a cell when RNApolymerase which binds the promoter sequence transcribes the codingsequence into mRNA which is then in turn translated into the proteinencoded by the coding sequence.

The term “nucleic acid” and “polynucleotide” refers to RNA or DNA thatis linear or branched, single or double stranded, or a hybrid thereof.The term also encompasses RNA/DNA hybrids. The following arenon-limiting examples of polynucleotides: a gene or gene fragment,exons, introns, mRNA, tRNA, rRNA, ribozymes, cDNA, recombinantpolynucleotides, branched polynucleotides, plasmids, vectors, isolatedDNA of any sequence, isolated RNA of any sequence, nucleic acid probesand primers. A polynucleotide may comprise modified nucleotides, such asmethylated nucleotides and nucleotide analogs, uracyl, other sugars andlinking groups such as fluororibose and thiolate, and nucleotidebranches. The sequence of nucleotides may be further modified afterpolymerization, such as by conjugation, with a labeling component. Othertypes of modifications included in this definition are caps,substitution of one or more of the naturally occurring nucleotides withan analog, and introduction of means for attaching the polynucleotide toproteins, metal ions, labeling components, other polynucleotides orsolid support. The polynucleotides can be obtained by chemical synthesisor derived from a microorganism.

The term “gene” is used broadly to refer to any segment ofpolynucleotide associated with a biological function. Thus, genesinclude introns and exons as in genomic sequence, or just the codingsequences as in cDNAs and/or the regulatory sequences required for theirexpression. For example, gene also refers to a nucleic acid fragmentthat expresses mRNA or functional RNA, or encodes a specific protein,and which includes regulatory sequences.

An “isolated” biological component (such as a nucleic acid or protein ororganelle) refers to a component that has been substantially separatedor purified away from other biological components in the cell of theorganism in which the component naturally occurs, for instance, otherchromosomal and extra-chromosomal DNA and RNA, proteins, and organelles.Nucleic acids and proteins that have been “isolated” include nucleicacids and proteins purified by standard purification methods. The termalso embraces nucleic acids and proteins prepared by recombinanttechnology as well as chemical synthesis.

The term “purified” as used herein does not require absolute purity;rather, it is intended as a relative term. Thus, for example, apartially purified polypeptide preparation is one in which thepolypeptide is more enriched than the polypeptide is in its naturalenvironment. That is the polypeptide is separated from cellularcomponents. By “substantially purified” is intended that such that atleast 60%, at least 70%, at least 80%, at least 90%, at least 95%, or atleast 98%, or more of the cellular components or materials have beenremoved. Likewise, a polypeptide may be partially purified. By“partially purified” is intended that less than 60% of the cellularcomponents or material is removed. The same applies to polynucleotides.The polypeptides disclosed herein can be purified by any of the meansknown in the art.

Variants include allelic variants. The term “allelic variant” refers toa polynucleotide or a polypeptide containing polymorphisms that lead tochanges in the amino acid sequences of a protein and that exist within anatural population (e.g., a virus species or variety). Such naturalallelic variations can typically result in 1-5% variance in apolynucleotide or a polypeptide. Allelic variants can be identified bysequencing the nucleic acid sequence of interest in a number ofdifferent species, which can be readily carried out by usinghybridization probes to identify the same gene genetic locus in thosespecies. Any and all such nucleic acid variations and resulting aminoacid polymorphisms or variations that are the result of natural allelicvariation and that do not alter the functional activity of gene ofinterest, are intended to be within the scope of the invention.

As used herein, the term “derivative” or “variant” refers to apolypeptide, or a nucleic acid encoding a polypeptide, that has one ormore conservative amino acid variations or other minor modificationssuch that (1) the corresponding polypeptide has substantially equivalentfunction when compared to the wild type polypeptide or (2) an antibodyraised against the polypeptide is immunoreactive with the wild-typepolypeptide. Such modifications may be deliberate, as by site-directedmutagenesis, or may be spontaneous. The term “variant” furthercontemplates deletions, additions and substitutions to the sequence, solong as the polypeptide functions to produce an immunological responseas defined herein.

The term “conservative variation” denotes the replacement of an aminoacid residue by another biologically similar residue, or the replacementof a nucleotide in a nucleic acid sequence such that the encoded aminoacid residue does not change or is another biologically similar residue.In this regard, particularly preferred substitutions will generally beconservative in nature, as described above.

A “vector” refers to a recombinant DNA or RNA plasmid or virus thatcomprises a heterologous polynucleotide to be delivered to a targetcell, either in vitro or in vivo. The heterologous polynucleotide maycomprise a sequence of interest for purposes of prevention or therapy,and may optionally be in the form of an expression cassette. As usedherein, a vector needs not be capable of replication in the ultimatetarget cell or subject. The term includes cloning vectors and viralvectors.

The term “recombinant” means a polynucleotide with semisynthetic, orsynthetic origin which either does not occur in nature or is linked toanother polynucleotide in an arrangement not found in nature.

“Heterologous” means derived from a genetically distinct entity from therest of the entity to which it is being compared. For example, apolynucleotide may be placed by genetic engineering techniques into aplasmid or vector derived from a different source, and is a heterologouspolynucleotide. A promoter removed from its native coding sequence andoperatively linked to a coding sequence other than the native sequenceis a heterologous promoter.

The polynucleotides of the invention may comprise additional sequences,such as additional encoding sequences within the same transcriptionunit, controlling elements such as promoters, ribosome binding sites,5′UTR, 3′UTR, transcription terminators, polyadenylation sites,additional transcription units under control of the same or a differentpromoter, sequences that permit cloning, expression, homologousrecombination, and transformation of a host cell, and any such constructas may be desirable to provide embodiments of this invention.

Operably Encodes or Associated. Operably encodes or operably associatedeach refer to the functional linkage between a promoter and nucleic acidsequence, wherein the promoter initiates transcription of RNAcorresponding to the DNA sequence. A heterologous DNA sequence is“operatively associated” with the promoter in a cell when RNA polymerasewhich binds the promoter sequence transcribes the coding sequence intomRNA which is then in turn translated into the protein encoded by thecoding sequence.

Promoter. A DNA sequence within a larger DNA sequence defining a site towhich RNA polymerase may bind and initiate transcription. A promoter mayinclude optional distal enhancer or repressor elements. The promoter maybe either homologous, i.e., occurring naturally to direct the expressionof the desired nucleic acid, or heterologous, I.e., occurring naturallyto direct the expression of a nucleic acid derived from a gene otherthan the desired nucleic acid. A promoter may be constitutive orinducible.

Vaccine. A vaccine is defined herein in its broad sense to refer to anytype of biological agent in an administrable form capable of stimulatinga protective immune response in an animal inoculated with the vaccine.

Embodiments

The present invention provides Recombinant Marek's disease virus (MDV),into which has been inserted via homologous recombination, a longterminal repeat (LTR) derived from a reticuloendotheliosis virus (REV).These recombinants are effective to elicit an immune response in anavian to Marek's disease virus without causing a significant degree ofpathogenicity in the avian. As used herein, “without causing asignificant degree of pathogenicity” is defined as no gross MD-specificlesions being observable with the naked eye in the inoculated/challengedavian, even in highly susceptible avians. In particular embodiments, theavians are chickens.

CVRM-2 was produced by authors of Reddy et al., as described therein,and as summarized in FIGS. 1 and 2 of this disclosure. Upon receipt ofthe CVRM2 sample, instant Applicants performed careful PCR-basedanalysis to confirm the identity/integrity of the virus isolate (FIG. 3presents the agarose gel resolution of the amplified products).Applicants found to their surprise the sample was not a clonal isolate,but was in fact a combined population of recombinant CVRM2 and theparental MDV strain. Applicants then performed the necessary plaquepurification to obtain a pure isolate consisting only of CVRM2 (and notthe parental MDV Rispens strain). For clarity, the new, clonal isolateis referred to as “RN1250” throughout this disclosure.

The recombinant MDV of this invention may be produced by modification ofMDV serotype 1 strain CVI988, or any of its clones or serially passagedstrains, which are collectively referred to herein as strains“CVI988/X”. Thus, as used herein CVI988/X includes, but is not limitedto, the previously described original low-passage strain, CVI988/Rispens(Rispens et al., 1972, Avian Dis., 16:106-125 and 126-138), strainCVI988 clone C (CVI988/C) (De Boer, U.S. Pat. No. 4,673,572, and De Boeret al., 1986, Avian Dis. 30:276-283), and CVI988/C/R6 (De Boer et al.,1988, Advances in Marek's Disease Research, pp. 405-413). The contentsof each of the above-mentioned publications/patents are incorporated byreference herein.

In an embodiment, the invention provides for a novel, recombinant,attenuated MDV strain, which is produced replacing a portion of thenative CVI988/X sequence with exogenous DNA, which comprises a longterminal repeat (LTR) sequence from a reticuloendotheliosis virus (REV).

In an embodiment, recombination of strain CVI988 was effected using MDVserotype 1 strain RM1 as a source of the exogenous LTRs (RM1 is arecombinant MDV into which REV LTRs had been integrated). As shown inFIG. 2, the REV LTR was excised from the purified RM1 viral DNA by Pac 1digestion and inserted into a shuttle vector, B40, prepared from a veryvirulent strain of Marek's disease virus, Md5. The resultant recombinantvector, B40-Pac, was used for insertion of the LTRs into the Marek'sdisease virus strain CVI988. To generate recombinant MDV with the LTRs,the purified viral DNA of MDV strain CVI988 was co-transfected intochicken or duck embryonic fibroblast (CEF or DEF) cells with NotI-digested recombinant vector. Recombinant viruses having the RM1 LTRsintegrated into their genome replicated more quickly than the parentalCVI988 strain. Without being bound by theory, it is believed that thisincreased rate of replication is the result of the insertion of thereticuloendotheliosis virus LTR into the genome of the MDV upstream ofthe ICP4 gene.

In another embodiment, the MDV CVI988/X may be modified via the additionof isolated reticuloendotheliosis virus LTRs from sources other than theRM1 strain. For instance, the insertion site of the LTR in the RM1strain of MDV has been shown to be between IRL and IRS of the genome(Jones et al., 1996, Retroviral insertional activation in a herpesvirus:transcriptional activation of US genes by an integrated long terminalrepeat in a MDV clone, J. Virology, 70(4):2460-2467). This correspondsapproximately to position 152,745 of the Md5 strains of Marek's diseasevirus. This region is located within a 1,704 base pair long EcoR1fragment (nucleotides 152, 198-153, 902) of serotype 1 Md5 (Tulman etal., 2000, The genome of a very virulent Marek's disease virus, J.Virology, 74d7):7980-7988). This 1,704 bp EcoR1 fragment can be clonedinto a plasmid vector lacking Drain restriction endonuclease site andused as a transfer vector for introduction of any LTR in to the MDVgenome. This EcoRX fragment has a unique DraIII restriction site located10 bp upstream of the LTR location in RM1. The LTRs can be inserted intothe DraIII site of the 1,704 base pair EcoR1 fragment to generate theLTR transfer vector, in order to generate recombinant MDV with LTRinsertions, the transfer vector should be linearized with EcoR1,extracted with phenol and chloroform and precipitated with ethanol.Co-transfection of the linearized transfer vector along with DNA fromany serotype 1 MDV strain into permissible cells in culture will resultin the introduction of LTR sequences into the MDV genome by homologousrecombination.

Reddy et al., supra, indicated the resulting recombinant virus (i.e. MDVwith a reticuloendotheliosis LTR) should “grow more rapidly than itscorresponding parental MDV strain, and thus there is no need for plaquepurification”. However, in view of Applicants' unexpected finding thatthe CVRM-2 sample was in fact a combination of recombinant MDV plus itscorresponding parental MDV, the skilled person is advised strongly toplaque purify all recombinant MDV envisioned by the instant disclosure.This is particularly important because a skilled person could bemistakenly encouraged to discard a potentially useful recombinant MDVafter obtaining results indicating immunization with the virus fails toprovide sufficient protection against a subsequent virulent MDVchallenge (please see Table 1 of Reddy et al., where CVRM-2 appears toprovide less than 80% protection).

In another embodiment, recombinant MDV having the RETV LTRs may beprepared from any MDV, including other CVI988/X strains, using thedeposited CVRM-2, provided the CVRM-2 is plaque purified to ensure thevirus is RN1250, and not a combination of RN1250 and the parental MDVstrain.

A variety of REV LTRs are suitable for use herein. Numerous suitablereticuloendotheliosis viral LTRs have been isolated and described, andinclude but are not limited to those described by Kost et al. (1993,Retrovirus insertion into herpesvirus: characterization of a Marek'sdisease virus harboring a solo LTR, Virology, 192:161-169), Ridgway(1992, REV LTR elements are efficient promoters in cells of variousspecies and tissue origin, including human lymphoid cells, Gene,121:213-218), Boerkoel and Kung [1992, Transcriptional interactionbetween retroviral long terminal repeats (LTRs): mechanism of 5′ LTRsuppression and 3′ LTR promoter activation of c-myc in avian B-celllymphomas, J. Virol., 66:4814-4823], Hippenmeyer and Krivi (1991, Geneexpression from heterologous promoters in a replication-defective avianretrovirus vector in quail cells, Poult. Sci., 70:982-92), Ridgway etal. (1989, Transient expression analysis of the reticuloendotheliosisvirus long terminal repeat element, Nucleic Acids Res., 17:3199-3215),Embretson and Temin (1987, Transcription from a spleen necrosis virus 5′long terminal repeat is suppressed in mouse cells, J. Virol.,61:3454-3462), Notani and Sauerbier (1987, Sequence instability in thelong terminal repeats of avian spleen necrosis virus andreticuloendotheliosis virus, J. Mol. Evol., 25:241-247), Robinson andGagnon (1986, Patterns of proviral insertion and deletion in avianleukosis virus-induced lymphomas, J. Virol., 57:28-36), and Ridgway etal., (1985, In vitro transcription analysis of the viral promoterinvolved in c-myc activation in chicken B lymphomas: detection andmapping of two RNA initiation sites within the reticuloendotheliosisvirus long terminal repeat, J. Virol., 54:161-170). The contents of eachof the publications referred to above are incorporated by referenceherein, in addition, numerous LTR sequences are available in GenBank andother genomic databases and can be synthesized by PCR using LTR specificprimers. The PCR amplified sequences can then be inserted in any of thetwo transfer vectors described as indicated above.

The REV LTR nucleic acid sequences disclosed herein, or theirbiologically functional equivalents, can be used in accordance with thepresent invention. The phrase “biologically functional equivalents” asused herein, denotes nucleic acid sequences exhibiting the same orsimilar biological activity/immunoprotective activity as theabove-mentioned reticuloendotheliosis viral LTR nucleic acid sequences(i.e., when introduced into the CVI988 MDV host in a functionallyoperable manner they elicit a protective immune response without causinga significant degree of pathogenicity in the chicken).

For example, the nucleic acid sequences described herein can be alteredby base substitutions, insertions, additions, or deletions to producebiologically functionally equivalent nucleic acids that retain promoteror enhancer activity.

The variants of the genomic DNAs or cDNAs (if obtained by RT-PCR fromRNA), contemplated herein should possess more than 75% homology,preferably more than 85% homology, and most preferably more than 95%homology, to the naturally occurring REV LTRs discussed herein.

The vaccine of the recombinant Marek's disease virus of the inventionmay be prepared as a cell-free preparation, or in the preferredembodiment, as a cell-associated preparation. A cell-associated vaccinecan be prepared directly from In vitro culture of the live viral agentsin a suitable growth medium, such as chicken embryo fibroblasts asdescribed by Witter (U.S. Pat. No. 4,895,718, the contents of which areincorporated by reference herein). Alternatively, to prepare cell-freevirus inocula, cells from infected host tissue or cell culture aresonicated or otherwise disrupted as previously described. The cellulardebris is removed by centrifugation and the centrifugate recovered asthe inoculum. Moreover, while the preferred vaccine is a viable virus,it is also envisioned that the vaccine may be prepared from the killedvirus or from immunogenic components separated from the virus, althoughsuch processing would incur significantly greater costs. For example, asubunit vaccine can be prepared by separating from the killed virus oneor more purified viral proteins identified as having immunogenicproperties.

The viral agent is prepared for administration by formulation in aneffective immunization dosage with a pharmaceutically acceptable carrieror diluent, such as physiological saline or tissue culture medium. Theexpression “effective immunization dosage” is defined as being thatamount which will induce immunity in a chicken against challenge by avirulent strain of Marek's disease virus, immunity is considered ashaving been induced in a population of chickens when the level ofprotection for the population is significantly higher than that of anunvaccinated control group (measured at a confidence level of at least80%, preferably measured at a confidence level of 95%). One measure ofthe level of protection is the protective index (Pi), which iscalculated as the incidence of MD in unvaccinated, MDV challengedcontrols minus the incidence of MD in vaccinated, MDV challenged groups,and the difference divided by the percent of Marek's disease inunvaccinated, MDV challenged controls, with the result multiplied by100.

Typically, the vaccine will contain at least about 200 PFU(plaque-forming units) of the virus, and preferably between about 2000and 5000 PFU. The vaccine can be effectively administered any time afterthe chicken attains immunocompetence, which is at about the 18th day ofincubation (3 days prehatch); but it is normally administered byinoculation within 24-48 hours after hatching. Alternatively, therecombinant viral DNA may be administered as a DNA vaccine as describedby Tischer et al. (2002, J. Gen. Virology, 83:2367-2376, the contents ofwhich are incorporated by reference herein).

Appropriate adjuvants as known in the art may also be included in thevaccine formulation. In many cases, the vaccinal efficacy can beenhanced by combining the recombinant Marek's disease viruses of theinvention with other viral agents into bivalent or polyvalent vaccines.

In another embodiment, the pharmaceutically or veterinarily acceptablecarrier, excipient, or vehicle may be a water-in-oil emulsion. In yetanother embodiment, the water-in-oil emulsion may be a water/oil/water(W/O/W) triple emulsion. In yet another embodiment, the pharmaceuticallyor veterinarily acceptable carrier, excipient, or vehicle may be anoil-in-water emulsion.

Methods of Use and Article of Manufacture

The present invention includes the following method embodiments. In anembodiment, a method of vaccinating an avian comprising administering acomposition comprising a Marek's Disease virus (MDV).

In one embodiment of the invention, a prime-boost regimen can beemployed, which is comprised of at least one primary administration andat least one booster administration using at least one commonpolypeptide, antigen, epitope or immunogen. Typically the immunologicalcomposition or vaccine used in primary administration is different innature from those used as a booster. However, it is noted that the samecomposition can be used as the primary administration and the boosteradministration. This administration protocol is called “prime-boost”.

A prime-boost regimen comprises at least one prime-administration and atleast one boost administration using at least one common polypeptideand/or variants or fragments thereof. The vaccine used inprime-administration may be different in nature from those used as alater booster vaccine. The prime-administration may comprise one or moreadministrations. Similarly, the boost administration may comprise one ormore administrations.

The dose volume of compositions is generally between about 0.1 to about2.0 ml, between about 0.1 to about 1.0 ml, and between about 0.5 ml toabout 1.0 ml.

It should be understood by one of skill in the art that the disclosureherein is provided by way of example and the present invention is notlimited thereto. From the disclosure herein and the knowledge in theart, the skilled artisan can determine the number of administrations,the administration route, and the doses to be used for each injectionprotocol, without any undue experimentation.

The present invention contemplates at least one administration to ananimal of an efficient amount of the therapeutic composition madeaccording to the invention. The animal may be male, female, pregnantfemale and newborn. This administration may be via various routesincluding, but not limited to, intramuscular (IM), intradermal (ID) orsubcutaneous (SC) injection or via intranasal or oral administration.The therapeutic composition according to the invention can also beadministered by a needleless apparatus (as, for example with a Pigjet,Dermojet, Biojector, Avijet (Merial, GA, USA), Vetjet or Vitajetapparatus (Bioject, Oreg., USA)). Another approach to administeringplasmid compositions is to use electroporation (see, e.g. Tollefsen etal., 2002; Tollefsen et al., 2003; Babiuk et al., 2002; PCT ApplicationNo. WO99/01158). In another embodiment, the therapeutic composition isdelivered to the animal by gene gun or gold particle bombardment. In anadvantageous embodiment, the animal is a dog, ferret or seal.

Another embodiment of the invention is a kit for performing a method ofeliciting or inducing an immunological or protective response againstMDV in an animal comprising a recombinant MDV immunological compositionor vaccine and instructions for performing the method of delivery in aneffective amount for eliciting an immune response in the animal.

In an embodiment, the subject matter disclosed herein is directed to akit for performing a method of eliciting or inducing an immune responsewhich may comprise any one of the recombinant MDV compositions orvaccines and instructions for performing the method.

Other cytokines that may be used in the present invention include, butare not limited to, granulocyte colony stimulating factor (G-CSF),granulocyte/macrophage colony stimulating factor (GM-CSF), interferon α(IFNγ), interferon β (IFNβ), interferon γ, (IFNγ), interleukin-1α(IL-1α), interleukin-1β (IL-1β), interleukin-2 (IL-2), interleukin-3(IL-3), interleukin-4 (IL-4), interleukin-5 (IL-5), interleukin-6(IL-6), interleukin-7 (IL-7), interleukin-8 (IL-8), interleukin-9(IL-9), interleukin-10 (IL-10), interleukin-11 (IL-11), interleukin-12(IL-12), tumor necrosis factor α (TNFα), tumor necrosis factor β (TNFβ),and transforming growth factor β (TGFβ). It is understood that cytokinescan be co-administered and/or sequentially administered with theimmunological or vaccine composition of the present invention. Thus, forinstance, the vaccine of the instant invention can also contain anexogenous nucleic acid molecule that expresses in vivo a suitablecytokine, e.g., a cytokine matched to this host to be vaccinated or inwhich an immunological response is to be elicited (for instance, anavian cytokine for preparations to be administered to avians).

The invention will now be further described by way of the followingnon-limiting examples.

Example 1 Efficacy of Marek's Disease Virus (MDV), SR-1 Strains, CVRM-2RN1250 and RMI CN32399, and Rispens CN32553

A critical aspect of the instant invention is that after havingcompleted the study outlined in this Example, inventors later determinedthe CVRM-2 MDV was not a clonal recombinant MDV, but in fact was a mixedpopulation of RN1250 recombinant MDV and parental Rispens MDV. Thus, nowthat inventors have produced a stable, clonal RN1250 MDV, which virus'strong efficacy as a vaccine is demonstrated in later Examples, askilled person will not be surprised by the broad (and unacceptable)range of protection (37-86%) apparently provided by the CVRM-2 MDV inthis preliminary study.

Objective.

To evaluate and compare the efficacy of three experimental MDV SR-1 andRispens strains to a commercial vaccine product (Rismavac®-Intervet,Inc.) in SPF Chickens using an early challenge with MDV T. King.

Materials/Methods.

One hundred fifty one-day-old SPF chicks (SPAFAS flock W-42) wererandomly assigned to five different groups of thirty birds each (Groups1-5). Each treatment group was then randomly assigned to isolationunits. The chicks in Group 1 used as the non-vaccinated, challengedcontrols were placed into negative pressure isolation units first,fifteen birds per unit. Each remaining group (Groups 2-5) were thensubcutaneously (SQ) vaccinated with 0.2 ml per bird with either IntervetRispens Rismavac®; MDV SR-1 RMI CN32399 P₄; MDV SR-1 CVRM-2 RN1250 P₄;or MDV Rispens CN32553 P₄. The SQ vaccinated birds in Groups 2-5 werealso placed in negative pressure isolation units, 15 birds per unit.Four days later on Study Day 4, the birds in Groups 1-5 were challengedindividually with MDV T. King, diluted 1:500 (protocol 02-085 02 January3). Birds were challenged intraperitoneally (IP), with 0.2 ml per bird.Birds were observed for 49 days post-challenge. Any bird that died priorto Study Day 53 was necropsied and examined for MDV lesions. On Day 53,all remaining birds were terminated, necropsied and examined for MDVlesions.

Results.

The experimental MDV SR-1 and Rispens strains provided protection ratesof 37-86% against the early MDV T. King challenge. Intervet's Rismavac®provided 83% protection. The challenge was validated with thenon-vaccinated, challenged control birds showing MD incidence of 97%.

Example 2 Efficacy of MDV, SR-1, RN1250 Vaccine in Commercial BroilersUsing a Shedder Challenge Model

Materials & Methods:

Seventy-six (76) one-day-old commercial broilers obtained from Harrisonpoultry flock 1-1 were randomized into three different colony houses andfour different groups as follows: Group 1: 13 birds; Group 2: 13 birds;Group 3: 25 birds; and Group 4: 25 birds (one house was split into twosides in order to create pen one and pen two). After randomizing, thebirds were banded in the nape of the neck for identification and thenwere inoculated with 0.2 ml per bird intraperitoneally (IP) with vvMDVT. King challenge, diluted 1:500. After challenging, the birds wereplaced into their respective colony house with 25-26 birds per house.These birds remained in the colony houses for 14 days prior to theplacement of MDV vaccinated and non-vaccinated, contact control birds.Fourteen days after placement of the shedder birds, 304 one-day-oldcommercial broilers were randomized as follows: two groups with 75 birdseach (Groups 3 and 4) and two groups with 38 and 40 birds each (Groups 1and 2), to serve as vaccinates; in addition, two groups of 25 birds andtwo with 13 birds were used to serve as non-vaccinated, contact controlbirds for Groups 1-4 (Group 1: 13 birds; Group 2: 13 birds; Group 3: 25birds; and Group 4: 25 birds). These contact control birds were bandedin the nape of the neck for identification and placed in the colonyhouses. The vaccinates were inoculated by the subcutaneous route (SQ)route (0.2 ml per bird) as follows: Group 1 was vaccinated with RN1250pre MSV, P6 (isolate I); Group 2 was vaccinated with RN1250 pre MSV, P7(isolate U); Group 3 was vaccinated with RN1250 pre MSV, P7 (isolate F);and Group 4 was vaccinated with Rismavac® serial 02760010, exp. 15 Apr.2012. After the inoculations, the vaccinates were placed in the samecolony houses where the non-vaccinated, contact control birds andshedders were placed previously. An additional fifty (50) non-inoculatedone-day-old commercial broilers were housed in isolation units, and werekept for 49 days in order to serve as a second group of contact controlsat a later day. All the vaccinated birds were banded in the nape of theneck eight days later with color coded, numbered bands. The sheddersremained in the colony houses for fifty days, and then all the survivorswere terminated and necropsied.

Clinical disease and mortality were monitored daily for 49 dayspost-vaccination. The spleens from any contact control bird that diedwere collected for virus isolation. On study day 53, the fortynon-inoculated commercial broilers that were housed in isolation units,in order to serve as the second group of contact controls, were placedin the colony houses, Groups 1-4, according to a randomization schedule,10 birds per group. These birds were banded with numbered bands beforeplacement for identification. The spleens from these birds were alsocollected for virus isolation in the event that mortality occurred. Theremaining vaccinates and the initial non-vaccinated, contact controlbirds were terminated and necropsied at the of the 49 day observationperiod on study day 63. Spleens and at least two feather follicles perbird were collected from 10 contact controls for virus isolation. Thesampled birds were determined by a randomization schedule. On study day83, the remaining contact control birds that were added to the groups onstudy day 53 (second group), were terminated. Spleens and at least twofeather follicles per bird were collected from these contact controlbirds for virus isolation.

TABLE 1 Number of birds surviving to five day of age # survivors to 5days of age/ Regime Group Name total # birds Vaccinated 1 RN1250 pre MSV(I) 38/38 Vaccinated 2 RN1250 pre MSV (U) 40/40 Vaccinated 3 RN1250 preMSV (F) 73/75 Vaccinated 4 Rismavac ® 73/75 Contact control 1 RN1250 preMSV (I) 12/13 Contact control 2 RN1250 pre MSV (U) 13/13 Contact control3 RN1250 pre MSV (F) 22/25 Contact control 4 Rismavac ® 23/25

TABLE 2 Protective indexes against the wMDV T. King challenge #infected/ total # Percent Protective Regime Gp Name birds InfectedIndex⁵ Vaccinated 1 RN1250 pre MSV (I)¹ 2/38 5.26% 87.4 (81.2)⁶Vaccinated 2 RN1250 pre MSV (U)² 6/40 15.0%  2.6 (46.4)⁶ Vaccinated 3RN1250 pre MSV (F)³ 6/73 8.22% 84.9 Vaccinated 4 Rismavac ®⁴ 15/73 20.5% 63.7 Shedders 1 RN1250 pre MSV (I) 9/12 75.0% N/A Shedders 2RN1250 pre MSV (U) 11/12  91.7% N/A Shedders 3 RN1250 pre MSV (F) 20/25 80.0% N/A Shedders 4 Rismavac ® 22/24  91.7% N/A Contact control 1RN1250 pre MSV (I) 5/12 41.7% N/A Contact control 2 RN1250 pre MSV (U)2/13 15.4% N/A Contact control 3 RN1250 pre MSV (F) 12/22  54.5% N/AContact control 4 Rismavac ® 13/23  56.5% N/A 2^(nd) controls 1 RN1250pre MSV (I) 9/10 90.0% N/A 2^(nd) controls 2 RN1250 pre MSV (U) 8/1080.0% N/A 2^(nd) controls 3 RN1250 pre MSV (F) 9/10 90.0% N/A 2^(nd)controls 4 Rismavac ® 8/10 80.0% N/A ¹RN1250 (I) vaccine titer: 3600pfu/0.2 ml. ²RN1250 (U) vaccine titer: 3144 pfu/0.2 ml. ³RN1250 (F)vaccine titer: 3710 pfu/0.2 ml. ⁴Rismavac ® vaccine titer: 2328 pfu/0.2ml. ⁵Protective index: Percentage of non-vaccinated contacts with MDlesions (challenge controls) − Percentage of vaccinated chickens with MDlesions/Percentage of non-vaccinated contacts with MD lesions (challengecontrols). ⁶Protective index considering the percentage ofnon-vaccinated contact controls with MD lesions in Colony House 10,Groups 1 and 2, as only one group (28% or 7/25).

Conclusion. The Marek's Disease Vaccine, Serotype 1, RN 1250 Vaccine,pre MSV (I) and (F), were more efficacious than Intervet's Rismavac® incommercial broilers using MDV T. King in a shedder challenge model

Example 3 Safety MDV RN1250 (X+5) in SPF One-Day-Old Chicks

Two hundred-fifty, one-day-old, SPF chickens were randomly assigned tofive treatment groups, 50 chicks per group, as well as, randomlyassigned to the negative pressure isolation units used for housing. Onehundred chicks were designated as vaccinates and were identified asGroups 1 and 2. One hundred chicks were designated as thesham-vaccinated, contact controls of Groups 1 and 2 (Groups 4 and 5,respectively); the remaining sham-vaccinated chicks (50) were identifiedas Group 3 to serve as the negative controls. After randomizing, thesham-vaccinated, contact controls and sham-vaccinated, negative controlswere wing-banded with numbered bands for identification and were placedinto their respective units (7-10 birds per unit). The sham-vaccinatedbirds were inoculated with Marek's Disease diluent, 0.2 ml per bird. Thechicks designated as vaccinates were subcutaneously (SQ) inoculated witheither: RN1250 (X+5) (Group 1) or Rismavac® (Group 2), 0.2 ml per bird(˜5000-6500 plaque-forming units (pfu) per dose). After theinoculations, the vaccinates were placed into their assigned units (7-10birds per unit) with their sham-vaccinated, contact controls which hadbeen previously housed, for a total of 15 to 20 chicks per unit.

TABLE 3 Study Design VACCINATION* NUMBER of GROUP VACCINE ROUTE BIRDS 1MDVSR-1 SQ 50 RN1250 2 MDVSR-1 SQ 50 Rismavac ® 3 Sham-vaccinated SQ 50(Negative Controls) 4 Sham-vaccinated Contact SQ 50 Controls for MDVSR-1 RN1250 vaccinated birds 5 Sham-vaccinated Contact SQ 50 Controlsfor MDV SR-1 Rismavac ® vaccinated birds

On Study Day 7, organ samples (bursa, thymus and spleen) from five birdsbelonging to each group, Groups 1-5, were chosen for harvesting andfixed in 10% buffered formalin for histopathology. The birds, 2-3 birdsper unit, were selected according to a randomization schedule. On StudyDay 14, body weights and organ weights (bursa, thymus and spleen) of 15birds from each group, Groups 1-5, were collected. Organ samples (bursa,thymus and spleen) from five birds in each group (Groups 1-5) wereharvested and fixed in 10% buffered formalin. Two to three birds perunit were used for the weighing and organ harvest. The birds wereselected according to a randomization schedule. The procedure describedon Study Day 14, was repeated on Day 28, and on Day 49 with theremaining birds.

Results.

Organs Atrophy RN1250. The bursa ratios of birds vaccinated with MDVSR-1 RN1250 vaccine and the ratios of the sham-vaccinated, contactscontrols were not significantly different from that of thesham-vaccinated, negative control birds, except that on Day 28, thebursa ratio of the contact controls for MDVSR-1 RN1250 was significantlyhigher than that of the negative controls (p≧0.0111). The thymus ratiosof birds vaccinated with MDVSR-1 RN1250 vaccine and the ratios of thesham-vaccinated, contacts controls were not significantly different fromthat of the sham-vaccinated, negative control birds (p≧0.7265). Finally,the spleen ratios of birds vaccinated with MDVSR-1 RN1250 vaccine andthe ratios of the sham-vaccinated, contacts controls were notsignificantly different from that of the sham-vaccinated, negativecontrol birds (p≧0.5286).

Organs Atrophy Rismavac®

The bursa ratios of birds vaccinated with MDVSR-1 Rismavac®-vaccine andthe ratios of the sham-vaccinated, contacts controls were notsignificantly different from that of the sham-vaccinated, negativecontrol birds (p≧0.0581). The thymus ratios of birds vaccinated withMDVSR-1 Rismavac®-vaccine and the ratios of the sham-vaccinated,contacts controls were not significantly different from that of thesham-vaccinated, negative control birds (p≧0.4004). The spleen ratio ofbirds vaccinated with MDVSR-1 Rismavac®-vaccine was significantlygreater than the spleen ratio of the sham-vaccinated, negative controlson Day 14 with p-value=0.0141; and the spleen ratios of birds ofsham-vaccinated contract controls was significantly greater than that ofthe sham-vaccinated, negative controls on Day 28 with p-value <0.0001.The ratios were not significantly different on any other days (p≧0.5558)Histological Examination Results from the histological examination didnot shown evidence of atrophy in the bursa, thymus or spleen of thebirds inoculated with the Marek's Disease SR-1 RN1250 or Rismavac®.

Conclusion.

Under the conditions of this trial the MDV SR-1, RN1250, X+5, was safewhen administered SQ, as evaluated by thymic, bursa and/or spleenatrophy.

Example 4 Dissemination of MDV, SR-1, RN1250 in SPF One-Day-Old Chicks

Objective.

To evaluate the dissemination of the MDV, SR-1, RN1250 experimentalvaccine (X+5) in one-day-old specific-pathogen-free (SPF) chickens whenadministered SQ and whether it would shed and spread to non-vaccinatedcontacts.

Materials & Methods.

One hundred twenty (120) one-day-old SPF chickens were randomized intothree different treatment groups and six units with each unit containing15 vaccinates and five contacts. The birds randomized as contacts werebanded in the nape of the neck with colored numbered bands per therandomization schedule and then placed into their respective units. Thebirds randomized as vaccinates were vaccinated with either MDV RN1250X+5 or Rispens. The experimental and commercial vaccines were diluted inMarek's diluent to yield approximately 100,000 pfu per dose(approximately 17× the expected field dose) administered by the SQroute. After vaccination, the birds were placed into their respectiveunits along with the contacts that were previously placed. Thevaccinated birds were banded at eight days of age in the nape of theneck with colored numbered bands according to a randomization schedule.Personnel involved with clinical assessments during the study were notpresent for the banding of the contacts or vaccinates nor did theyperform any clinical observations during this time period in order tomaintain blinding.

TABLE 4 Study Groups NUMBER of BIRDS* VACCINATION Non- ROUTE/ vaccinatedGP VACCINE VOLUME Vaccinated contacts 1 MDV SR-1 SQ/0.2 ml 30 10 RN1250per bird 2 MDV SR-1 SQ/0.2 ml 30 10 Rispens vaccine per bird 3Sham-vaccinated SQ/0.2 ml 30 10 Negative Controls per bird

At two weeks post-vaccination, tracheal and cloacal swabs were taken forvirus recovery from all vaccinated birds. The swabs were pooled bygroup, with five tracheal or cloacal swabs per swab tube containing 5 mlof SPGA stabilizer. Primary feather follicles were collected for virusrecovery from two vaccinated birds in each group, collecting two tothree feather samples per bird. The two birds per group for featherfollicle sampling were selected according to a randomization scheduleand were kept alive after sampling. All samples were taken to MerialSelect's analytical department for processing. The same samplingprocedure was repeated two times at seven day intervals.

On study day 21, in addition to tracheal and cloacal swab sampling andfeather follicle sampling, five vaccinated and non-vaccinated contactsper group (selected according to a randomization schedule) wereterminated and necropsied, with spleens were individually harvested.Virus isolation was attempted only on the spleens harvested from thenon-vaccinated contacts (vaccinated birds were harvested to maintainblinding). On the last day of the study (Day 49), all the remainingbirds were euthanized and necropsied and the study was terminated.Spleens from the remaining five vaccinated and non-vaccinated contactsper group were individually harvested. Virus isolation was onlyattempted on the spleens harvested from the non-vaccinated contacts(vaccinated birds were harvested to maintain blinding).

Results.

Group one MDV SR-1 RN1250 X+5 arithmetic mean titer (AMT) was 94,160pfu/0.2 ml dose. Group two MDV SR-1 Rispens was 51,800 pfu/0.2 ml dose.All tracheal and cloacal swabs were negative for virus recovery.

Group 1 MDV SR-1 RN1250: All of the samples tested for this group werenegative for virus recovery from feather follicles.

Group 2 MDV SR-1 Rispens: On day 21, feather follicles from both of thebirds sampled were positive for Marek's disease cytopathic effect (MDCPE). On days 14 and 28 feather follicles from all birds sampled werenegative.

Group 3 Sham-vaccinated negative controls: All of the feather folliclesamples tested were negative for virus recovery from feather.

There was no virus recovered from any of the spleens harvested from thecontacts birds at Days 21 and 49. The samples from day 49 were initiallycontaminated but were tested again using PCR and were all negative.Frozen buffy coat material from these same samples was also re-platedand all of these samples were confirmed negative.

Conclusion.

Under the conditions of this study, birds vaccinated SQ with the MDV,SR-1, RN1250 experimental vaccine showed similar dissemination patternsto birds vaccinated SQ with the commercially available MDV, SR-1 Rispensvaccine. There was no recovery of virus or evidence of clinical diseasein non-vaccinated birds that were in contact with birds vaccinated withthe MDV RN1250 experimental vaccine.

Example 5 Evaluation of the Efficacy of MDV, S1, RN1250 Vaccine (X+5)Administered to Day-Old Chicks Against vvMDV, RB1B Virus

Materials & Methods.

Two hundred eighty (280) one-day-old SPF chicks were randomized intoeight different groups and 24 different isolation units according to arandomization schedule (11-12 birds per unit; 35 birds per treatment).After the randomization, the birds in Groups 6 and 7, thesham-vaccinated, challenged and sham-vaccinated/sham challenged negativecontrols, were sham vaccinated with Marek's vaccine diluent and wereplaced into their designated units according to the randomizationschedule. The remaining birds were then vaccinated with MDV SR-1 RN1250,at either 287, 578, 736, 1085, or 1392 plaque forming units (pfu's) perbird dose, or with MDV SR-3 HVT, 1728 pfu's per bird dose. The birdswere vaccinated subcutaneously (SQ) with 0.2 ml per chick. After thevaccinations, each vaccinated group was placed into its designated unitaccording to the randomization schedule. On study day 4, the birds inGroup 7 were sham challenged with Marek's vaccine diluent and Groups 1-6and 8 were challenged with vvMDV RB1B, by the intraperitoneal (IP)route, 0.2 ml per bird. The birds were observed daily for 45 dayspost-challenge for any unfavorable reactions to the challenge,particularly death or depression. On study day 49, the birds wereterminated and necropsied to examine for gross lesions associated withMarek's disease.

Results.

The prevented fraction rates against the vvMDV RB1B challenge in Groups1-5 had a range of 0.84 to 0.94. The prevented fraction in Group 8, theHVT vaccinated group, was 0.73. The incidence of MDV in the shamvaccinated, challenge control Group 6 was 91.2%. The birds in Group 7,the sham vaccinated, sham challenged negative controls, remained free ofMDV lesions throughout the study.

Conclusion.

The MDV vaccine, Serotype-1, Live Virus, RN1250 Experimental Vaccine(X+5) administered subcutaneously (SQ) to day-old SPF chickens wasefficacious at 287 plaque forming units per dose using the RB1B virus asthe challenge.

TABLE 5 Dose Response Efficacy Summary # infected/total % % Gp Gp GroupName Actual dose # of birds Protected infect. 1 B MDV RN1250 (X + 5)vaccine 250 pfu 287.2 pfu/0.2 ml 5/34 85.3% 14.7% (AMT) 2 H MDV RN1250(X + 5) vaccine 500 pfu 578.4 pfu/0.2 ml 4/34 88.2% 11.8% (AMT) 3 C MDVRN1250 (X + 5) vaccine 750 pfu 736 pfu/0.2 ml 2/34 94.1% 5.9% (AMT) 4 AMDV RN1250 (X + 5) vaccine 1000 pfu 1084.8 3/35 91.4% 8.6% pfu/0.2 ml(AMT) 5 D MDV RN1250 (X + 5) vaccine 1500 pfu 1392 pfu/0.2 ml 4/34 88.2%11.8% (AMT) 6 E Sham vaccinated/Challenge Controls 31/34  N/A 91.2% (8.8%*) 7 G Sham vaccinated/Sham Challenge 0/35 N/A 0.0% NegativeControls 8 F MDV HVT Release titer 1725 pfu/0.2 ml 8/33 75.8% 24.2%(AMT)

Having thus described in detail preferred embodiments of the presentinvention, it is to be understood that the invention defined by theabove paragraphs is not to be limited to particular details set forth inthe above description as many apparent variations thereof are possiblewithout departing from the spirit or scope of the present invention.

What is claimed is:
 1. A method of eliciting in an avian animal a safe and protective immune response against Marek's Disease virus (MDV) comprising administering to said animal a therapeutically effective amount of a composition comprising a viral agent comprising a recombinant Marek's disease virus stably transformed with a foreign DNA construct, comprising a long terminal repeat (LTR) sequence of a reticuloendotheliosis virus, thereby eliciting the protective immune response.
 2. The method of claim 1 wherein the composition further comprises a veterinarily or pharmaceutically acceptable carrier or diluent.
 3. The method of claim 1 or 2 wherein the MDV is a CVI988/X MDV.
 4. The method of claim 3 wherein the CVI988/X MDV is CVI988.
 5. The method of claim 1 or 2 wherein the avian animal is a chicken.
 6. The method of claim 1 or 2 wherein the LTR is as set forth in SEQ ID NO:2.
 7. The method of claim 1 or 2 wherein the LTR sequence comprises a Pac I excised-DNA segment from an MDV having all of the identifying characteristics of the strain deposited at ATCC under the accession number PTA-4945.
 8. The method of claim 1 or 2 wherein the LTR sequence is inserted 5′ of the ICP4 gene of said MDV.
 9. The method of claim 1 or 2 wherein the viral agent is effective to elicit an immune response in an avian against MDV without causing a significant degree of pathogenicity in said avian.
 10. The method of claim 1 or 2 wherein the viral agent is cell-associated.
 11. A method of making a viral agent effective for protecting an avian against Marek's disease comprising transforming an MDV strain CVI988 with a foreign DNA construct which comprises a LTR sequence of a reticuloendotheliosis virus.
 12. A Marek's disease virus (MDV) stably transformed with a foreign DNA construct, comprising a long terminal repeat (LTR) sequence of a reticuloendotheliosis virus.
 13. The MDV of claim 12 comprising a nucleotide having at least 90% homology as the sequence as set forth in SEQ ID NO:2.
 14. The MDV of claim 13 wherein the nucleotide has the sequence as set forth in SEQ ID NO:2.
 15. An immunological composition comprising the MDV of any one of claims 12-14.
 16. The composition of claim 15 wherein the MDV is a clonal virus, and not a mixed population of parental and recombinant virus.
 17. The composition of claim 16 which is a vaccine composition.
 18. The composition of claim 17 which provides a protection rate of at least 85%.
 19. The composition of claim 18 which provides a protection rate of at least 90%.
 20. The composition of claim 19 which provides a protection rate of at least 95%.
 21. An isolated cell stably transformed with the MDV of claim 13 or
 14. 22. The isolated cell of claim 15, wherein the cell is either a chicken or duck embryo fibroblast (CEF, DEF) cell. 